MFR PAPER 1145 Gilbert B. Pauley was with the Pathobiology Investigations sec­ tion of the Middle Atlantic Coastal Fisheries Center, National Marine Fisheries Service, NOAA, Oxford, Seru m Changes in the Blue , MD 21654. He is now with the Washington Cooperative Fishery , Associated With Unit, College of Fisheries, Univer­ Paramoeba perniciosa, the Causative sity of Washington, Seattle, WA 98195. Martin W. Newman is with Agent of Gray Crab Disease the Pathobiology Investigations section of the Middle Atlantic Coastal Fisheries Center, National Marine Fisheries Service, NOAA, GILBERT B. PAULEY, MARTIN W. NEWMAN, and EDITH GOULD Oxford, MD 21654. Edith Gould is with the Experimental Biology In­ ABSTRACT-Hemolymph from healthyblue (Callinectes sapidus) was vestigations section of the Middle Atlantic Coastal Fisheries Center, compared with that ofanimals infected with Paramoeba perniciosa, the causa­ National Marine Fisheries Service, tive agent of gray crab disease. There was a significant difference (P = 0.01) NOAA, Milford, CT 06460. between normal and gray crab sera in both the protein and the glucose values. Analysis of infected blue crab serum by both immunoelectrophoresis and Newman and Ward (1973). Hemolymph acrylamide gel electrophoresis showed altered patterns of total protein and from healthy crabs, placed in a test hemocyanin that could be accurately correlated with the severity of the dis­ tube, was allowed to clot around an ap­ ease. plicator stick after which the clot was easily removed. Because the hemo­ A previously unknown disease of the MATERIALS AND METHODS lymph of most crabs infected with P. commercially important blue crab perniciosa does not clot, it was centri­ The crabs, C. sapidlls, used in this (Callinectes sapidlls) which killed 20-30 fuged at 5,000 rpm for 10 min at 4°C, percent of the animals held in commer­ study were obtained from Chincoteague to remove cellular debris. The resultant Bay, near Greenbackville, Va. Crab cial shedding tanks was described by sera were passed through Swinnex-25 sampling began on 26 May 1972, with Sprague and Beckett (1966). When millipore filters (0.45 fJ pore size) into six subsequent samplings during June viewed ventrally, moribund and dead sterile tubes, divided into 1.0 ml lots, 1972. The sample size varied from 4 to specimens had a translucent gray ap­ dated, and frozen at -25°C. Sera from 26 animals, with a total of 84 animals pearance, hence the name "gray crab individual crabs were always kept sepa­ being examined. Control sera for im­ disease." Sprague and Beckett (1968) rate. munoelectrophoresis were obtained ascertained that the etiological agent of Two rabbits were used for each anti­ from uninfected Chesapeake Bay crabs, the disease was an , subse­ serum produced. Freund's complete ad­ taken near Oxford, Md. quently named Paramoeba perniciosa. juvant (Difco) was mixed with an equal The posterior portion of the carapace The amoebae cause lysis of muscle amount ofcrab serum and administered was swabbed with 70 percent ethanol and blood cells and may completely re­ weekly for 5 weeks as 1.0 ml subcutane­ before withdrawing hemolymph from place the normal blood cells in the ous and 2.0 ml intramuscular injections the pericardial cavity with a sterile 5.0 hemolymph prior to death of the crabs in the rabbits. Two weeks after the final or 10.0 ml syringe and 20-gauge J1I2-inch (Sprague et aI., 1969; Sawyer et aI., injection, antiserum was collected by needle. A drop of hemolymph from 1970). Epizootiological studies have cardiac puncture, filter-sterilized, and each animal was placed on a microscope shown the disease is ephemeral, occur­ frozen at -25°C until used. Antisera slide and spread evenly in a thin film. ring each summer in the latter part of were made against the sera from 13 in­ This was allowed to air dry for 1-5 min June, and has a restricted range from fected crabs from Chincoteague Bay and then placed in 10 percent neutral­ Maryland to North Carolina (Sawyer, and from two normal male and two buffered formalin seawater. Smears 1969; Newman and Ward, 1973). normal female crabs from Chesapeake prepared from gray-appearing crabs In another commercially important Bay. Immunoelectrophoresis and stain­ were fixed in separate containers to crustacean, Homarus americanus, it ing were carried out as described by avoid the possibility ofamoebae floating has been found that bacteria, Gaffkya Pauley (1974). offthe slide and contaminating adjacent homari, cause serious alterations in The total protein concentration in the hemolymph chemistry (Stewart et aI., slides. Hemolymph smears were serum of 66 crabs was analyzed by the stained for 10 min with Giemsa l stain 1969; Stewart and Cornick, 1972). F olin-Ciocalteau method (Lowry et aI., diluted 1:5 with phosphate buffer, pH Therefore, a study was undertaken to 1951). Serum glucose was determined 6.8, and examined for the presence ofP. determine whether the hemolymph for 60 crabs by the method of Hultman perniciosa. The slides were then scored from blue crabs infected with P. (1959). Because ofthe small sample size as normal, light, moderate, or heavy in­ perniciosa differed from that of normal of some groups and because they were fection, based on the relative number of crabs. This paper reports our findings not homogeneous by analysis of the amoebae present as described by on the changes observed in the serum variance using the F distribution (Sne­ glucose, serum protein, and serum elec­ decor, 1956), no statistical tests for I Reference 10 lrade names does nol imply en­ trophoretic patterns in infected C. dorsemenl by the National Marine Fisheries Ser­ significance were attempted for the var­ sapidus. vice, NOAA. ious groups other than between pooled 34 normal and pooled heavily-infected Table 1.-Concentratlons of total protein and glucose In normal and heavily parasitized blue crab serum.

gray crab samples. The I-test for com­ Total protein (mg/mll Glucose (mg/l00 ml) paring the means of two randomized Sid. Std. Sid. Sid. groups of unequal size was used for No. Range Mean dey. error No. Range Mean dey. error statistical analysis ofthis data according Normal to the procedure outlined by Snedecor Males 11 25.0.67.0 45,4 15.1 4.5 11 10.5-66,4 34.7 18,4 5.5 Females 11 5.3·22,4 14.6 5.6 1.7 15 n~-74,4 24.4 18.6 4.8 (1956). Sponge 10 3.9-55,4 31.0 15.7 5.0 6 31.8·130.3 74,4 37.2 15.2 Aliquots (ca. 3 /.II) of serum from All 32 3.9-67.0 30.3 18.0 3.2 32 7.9-130.3 37.3 28.9 5.1 individual blue crabs were subjected to Gray Males 23 2.8·18.5 9,4 4.6 1.0 18 0.61.1 14.1 15.8 3.7 electrophoresis at 4°C on 7 percent ac­ Females 11 2.8·13.7 7,4 3.9 1.2 9 0-29.2 9,4 10,4 3.5 rylamide gel columns, pH 9. I, with All 34 2.8·18.5 8.8 4,4 0.8 28 0-61.1 11.1 14.1 2.7 sample and stacker gels of 3 percent acrylamide, pH 5.2. Electrode buffer 70- 1.30- was 0.005M Tris (hydroxymethyl 1.40- 60­ aminomethane) - 0.038 M Glycine, pH 1.10- 8.3. Running time was 60 min at I ~ 50- ~I.OO- , milliampere/column followed by 70-75 E'" E ;; 40- -;;. .90- min at 3 milliamperes/column with con­ ~ stant current. Both gel formulas and ~ .80- o 30- l)l 70- electrophoretic procedure are based on 8: O' g 60- the work of Davis (1964) and have been ...I 20- ...I' ;~' ~ ~ < ?;;f fully described by Gould and Medler ~ .50- 10- ~ (1970). o·40- Subsequent to electrophoresis, gels F- .:30- 0- .i were stained for total protein or for cop­ .20- ~ per. The stain used for total protein was j ::l , ! .. .10- $ L.... _ NORMAL GRAY Amido Schwartz 10 B (Buffalo Black), I 0- ...... J I~ percent in 7.5 percent acetic acid. Gels ... ~ .. '"z ...J .. Figure 1.-Graphlc presentation 01 the range, .. ...::l! f ::l .. ...::l! ::l were destained by passive diffusion in standard deviation, standard errOf, and mean of ::l! IL II> .. ::l! IL .. several changes of methanol-glacial total serum protein concentration In normal and NORMAL GRAY heavily parasitized blue crabs. acetic acid-distilled water (5: I:5) for a total ofabout 20 h. Hemocyanin sites on Figure 2.-Graphlc presentation 01 the range, normal and the gray crabs. Serum con­ standard deviation, standard error, and mean of . the gels were marked by a stain for cop­ centrations ofprotein and glucose cou Id total serum glucose concentration In normal and heavily parasitized blue crabs. per using an aqueous tetrazolium­ often be correlated with the intensity of cyanide solution (Gould and Karolus, infection (Table 2). In press), that proved to be faster and guishable (Fig. 5). Although no heavily more consistently reliable than the IMMUNOELECTROPHORESIS infected female crabs were analyzed by classic rubeanic acid stain (Horn and The normal immunoelectrophoretic this method, because of a shortage of Kerr, 1969). patterns of male and female crabs were hemolymph, there was never any doubt about a correct diagnosis with this BIOCHEMICAL ANALYSIS different (Figs. 3, 4). Infection of C. sapidus by P. perniciosa caused a pro­ method when hemolymph was tested Concentrations of total protein and gressive loss of serum protein as ob­ from moderately or heavily infected glucose in the serum of heavily infected served by immunoelectrophoresis, male crabs. and uninfected crabs used in this study which was apparently related to the ACRYLAMIDE GEL are listed in Table I. The range, stan­ severity of the infection (Figs. 3, 4). dard deviation, standard error, and There was some overlap in the appear­ ELECTROPHORESIS mean of these samples are graphically ance of the immunoelectrophoretic pat­ Typical electropherograms for male presented in Figures I and 2. The dis­ terns of normal and lightly infected and female blue crabs with varying de­ tribution of the values was skewed in animals which were often not distin- grees of P. perniciosa infection show some groups toward the lower values, such as the glucose of normal females, Table 2.-Blood glucose, blood protein. and degree ollnlectlon by Paramoeba pern/closa In the exemplar blue crab hemolymph used lor acrylamlde gel pherograms. where the value of the mean minus one Pherogram standard deviation exceeded the lower no. in Blood Blood State of infection limit of the range. As indicated in the figures Date of glucose protein observed in Giemsa 6 and 7 (mg/100 ml) (mgiml) Materials and Methods section, statis­ capture Sex stained blood smears tical tests of significance were per­ 1 6/30172 9 2.8 Heavy inlection 2 6/28/72 9 2.5 6.5 Moderate infection formed only between pooled normal 3 6/23/72 9 3.9 17.5 Light infection serum values and pooled heavily in­ 4 5/26/72 9 37.1 43.9 Normal fected gray crab values. There was a 5 6/28/72 " 0.0 2.8 Heavy infection 6 6/30172 < 5.2 7,4 Moderate infection significant difference (P = 0.0 I) in both 7 6/16/72

CD-L.-'_~ --"__ been observed in infected lobsters infected with G. homari. The reduction (Stewart et aI., 1969; Stewart and of glucose which we observed in C. Rabin, 1970). Diet alone is capable of sapidus may be attributed, at least in influencing crustacean hemocyte num­ part, to uptake and utilization by P. bers (Stewart et aI., 1967), and the pres­ perniciosa, resulting from the hosts' in­ ence of actively phagocytizing cells in ability to compete successfully for their invertebrates does not insure pathogen own nutrients, as in the case in lobsters destruction (Cornick and Stewart, 1968; infected with G. homari.

A

FIgure 4.-Immunoeleelrophoretlc panern. of male blua crabs. A show. a lightly Infacted male at the top. B shows a moderately Infected male at the top. C shows a heavily Infected male at the top. Figure 6.-Pherogram of female crab hemolymph Normal male patterns are on the bottom In all stained for copper. Heavy Infection - A; moderate cases. Central trough contains rabbit antiserum Infection -Bolight Infecllon - Co normal· D. Arrow against normal male crab serum. indicates migration direction.

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Figure 5.-Llghtly Infected mele at top .hows an Immunoelectrophoretic panern that Is the same as Figure 7.-Pherogram of male crab hemolymph the normal male at bonom. Rabbit antisarum stained for copper. Heavy Infeellon • A; moderate against normal male crab serum Is In central Infection - B; IIghtlnfecllon - C; normal - D. Arrow trough. Indicates migration dlrecllon. 36 methods would account for the failure of the infected crab hemolymph to clot. CD CD The secretion of powerful enzymes by the parasite would also account for the extensive cellular lysis observed in diseased animals. The rapid destruction of the slow HCy (8) during infection by P. perniciosa does not occur with Iyo­ trophic agents, such as urea, in which Figure 8.-Pherogram of female crab hemolymph case it is the fast HCy (Q) that are de­ slalned for lolal protein. Heavy infection - A; mod­ stroyed initially (Gould and Karolus, In erale Infection - B; IIghl infection - C; normal - D. Arrow indicates migration direction. press). This is further evidence that the amoebae, directly or indirectly, are al­ tering covalent honds of molecules within the host. Since hemocyanin is the oxygen binding and transporting molecule of crustaceans, the loss of hemocyanin indicates there probably is insufficient oxygen binding and trans­ port. Death, therefore, may be due to a

Figure 9.-Pherogram of male crab hemolymph combination of insufficient oxygen and slalned for tolal protein. Heavy Infection - A; mod­ nutrients. erate Infeclion - B; light Infection - C; normal - D. Arrow indicates migration dlrecllon. Minor electrophoretic changes were observed in infected lobsters (Stewart et aI., 1969), but these irregularities were not greatly pronounced at the time of death. Analysis of infected blue crab serum by both immunoelectrophoresis and acrylamide gel electrophoresis showed significantly altered patterns of total protein and hemocyanin that could be correlated with the severity of the disease in all but 4 of the 79 cases Normal total protein values obtained drastic, decline in serum prot.:in, which analyzed. Acrylamide gel protein pat­ in this study for C. sapidus serum is not regarded as a serious factor in the terns corroborate the results obtained (3.9-67.0 mglml, x = 30.3 mglml) are in deterioration of the animals' physiolog­ with immunoelectrophoresis (Fig. 10). close agreement with the normal values ical condition (Stewart et aI., 1969). In Copper electropherograms may also be observed in lubsters (Stewart et aI., blue crahs, the drop from 30.3 mglml to sensitive indicators ofinfection because 1967) but are somewhat lower than 8.8 mg/ml in infected animals is of the rapid disappearance of those values observed in the blue crab significant and probably accounts, in hemocyanin during the disease. Al­ by other investigators (Lynch and part, for the extremely weakened condi­ though electrophoretic analysis is prob­ Webb, 1973a). In lobsters infected with tion and rapid deterioration of crabs in­ ably not specific for this infection, we G. homari, there is a significant, but not fected wi th P. pcrniciosa . Stewart et al. recommend acrylamide gel electro­ (1907) have shown that protracted diet phoresis of the serum to determine the changes can cause severe serum protein degree of stress because of its relative 0· losses in the American lobster. How­ ease of performance and clarity of ever, in the infected blue crabs, diet results. change alone probably would not ac­ count for the large drop in serum pro­ tein, because the diseased crabs are LITERATURE CITED captured in baited crab pots within 24 h of examination, indicating that they Bang. F. B. 1970. Disease mechanisms in crusta­ 1 feed actively into the final stages of the cean and marine arthropods. Am. Fish. Soc. Spec. Pub!. 5:383-404. disease. The amoebae are probably Cornick. J. W., and J. E. Stewart. 1968. either pinocytosing serum proteins of Interaction of the pathogen Golf/cyo homor; with nalural defense mechanisms of Homorus ® the crab or secreting proteolytic en­ omer;conus. J. Fish. Res. Board Can. 25:695-709. Figure 10.-Pherogram of male crab hemolymph zymes which hydrolyze them into small Davis. B. J. 1964. Disc electrophoresis - II. stained lor total proleln. Hemolymph samples are peptides and amino acids, which may Method and application to human serum pro­ from the same three infected animals whose im­ teins. Ann. N.Y. Acad. Med. 121:404-427. munoelectrophoretic patterns are shown in Figure then be consumed by the parasite. The Florkin, M. 1960. Blood chemistry. In T. H. 4. Heavy Infection - A; moderate Infection - B; light loss of fibrinogen as part of the total Waterman (editor), The physiology of Crus­ Infection - C; normal- D. Arrow Indicates migrallon tacea, I: 143-159. Academic Press, N.Y. direction. serum protein by either of these Gould, E., and J. J. Karolus. In press. A new 37 stain for copper-protein complexes: Its use with caused by Paramoeba pemiciosa. J. Invertebr. and Virginia. J. Invertebr. PathoI. 8:287-289. crustacean hemocyanin. Anal. Biochem. Pathol. 22:329-334. _____. 1968. The nature of the etiological Gould, E., and M. J. Medler. 1970. Test 10 de­ Pauley, G. B. 1974. Comparison ofa natural ag­ agent of "gray crab" disease. J. Invertebr. termine whether shucked oysters have been fro­ glutinin in the hemolymph of the blue crab, PathoI. II :503. zen and thawed. J. Assoc. Of. Anal. Chem. Callillecles sapidus, with agglutinins of other in­ Sprague, V., R. L. Beckett, and T. K. Sawyer. 53: 1237-1241. vertebrates. Contemp. Top. Immunobiol. 1969. A new species of Paramoeba Horn. E. C., and M. S. Kerr. 1969. The hemo­ 4:241-260. (Amoebida, ) parasitic in the crab lymph proteins of the blue crab, Callillecles Pauley, G. B., S. M. Krassner, and F. A. Chap­ Callinecles sapidus. J. Invertebr. PathoI. sapidus-1. Hemocyanins and certain other man. 1971. Bacterial clearance in the California 14: 167-174. major protein constituents. Compo Biochem. sea hare, Aplysia cali/omica. J. Invertebr. Stewart, J. E., B. Arie, B. M. Zwicker, and J. R. Physiol. 29:493-508. Pathol. 18:227-239. Dingle. 1969. GaflKemia, a bacterial disease of Hultman. E. 1959. Rapid specific method for de­ Sawyer, T. K. 1969. Preliminary study on the the lobster, H omams americallus: Effects of the termination of aldosaccharides in body fluids. epizootiology and host-parasite relationship of pathogen, Gaffkya homari. on the physiology of Nature (Lond.) 183: 108-109. Paramoeba sp. in the blue crab, Callillecles the host. Can. J. Microbiol. 15:925-932. Lowry, O. H., N. J. Rosebrough, A. L. Farr, and sapidus. ProC. Natl. Shellfish. Assoc. 59:60-64. Stewart, J. E., and J. W. Cornick. 1972. Effects R. J. Randall. 1951. Protein measurement with Sawyer, T. K., R. Cox, and M. Higgenbottom. of Gaffkyo homari on glucose, total carbohy­ the Folin phenol reagent. J. BioI. Chern. 1970. Hemocyte values in healthy blue crabs, drates, and lactic acid of the hemolymph of the 193:265-275. Callillecles sapidus, and crabs infected with the lobster (Homarus americallus). Can. J. Mi­ Lynch, M. P., and K. L. Webb. 1973a. amoeba, Paramoeba pernieiosa. J. Invertebr. crobiol. 18:1511-1513. Variations in serum constituentsofthe blue crab, Pathol. 15:440-446. Stewart, J. E., J. W. Cornick, and J. R. Dingle. Callillecles sapidus: Total serum protein. Compo Sindermann, C. J. 1971. Internal defenses of 1967. An electronic method for counting lob­ Biochem. Physiol. 44A: 1237-1249. Crustacea: A review. Fish. Bull., U.S. ster (Homams americanus Milne Edwards) ____-,. 1973b. Variations in serum con­ 69:455-489. hemocytes and the influence ofdiet on hemocyte stituents of the blue crab, Callillecles sapidus: Snedecor, G. W. 1956. Statistical methods ap­ numbers and hemolymph proteins. Can. J. Zool. Glucose. Compo Biochem. Physiol. plied to experiments in agriculture and biology. 45:291-304. 45A: 127-139. 5th ed. Iowa State Univ. Press, Ames, 534 p. Stewart, J. E.. and H. Rabin. 1970. Gaffkemia, a Newman, M. W., and G. E. Ward, Jr. 1973. An Sprague, V., and R. L. Beckett. 1966. A disease bacterial disease of lobsters (genus H omams). epizootic of blue crabs, Callillecles sapidus, of blue crabs (Callilleeles sapidus) in Maryland Am. Fish. Soc. Spec. Publ. 5:431-439.

MFR Paper 1145. From Marine Fisheries Review, Vol. 37, Nos. 5-6, May.,}une 1975. Copies of this paper, in limited numbers, are available from 083, Technical Information Division, Environmental Science Infor­ mation Center, NOAA, Washington, DC 20235. Copies of Marine Fisheries Review are available from the Superintendent of Documents, U.S. Gov­ ernment Printing Office, Washington, DC 20402 for $1.10 each.

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